Project Summary/Abstract Periodontitis is an inflammatory disease of the periodontium caused by bacterial biofilms and affects approximately 65 million Americans. Periodontal pockets serve as bacterial reservoirs and create potential risks to develop systemic illness. Aggregatibacter actinomycetemcomitans, an oral colonizer in 20% of the population, is a causative agent of periodontitis and infective endocarditis. The increasing antibiotic-resistant strains and the limitation of developing new antibiotics demand alternative therapeutics to cure bacterial infections. Bacteriophage therapy is listed as the top alternative and has successfully treated several bacterial infections. Currently, no phage therapeutics has been pursued to treat infections caused by oral bacteria. Development of new phage therapeutics requires understanding phage infection and replication in vivo. This knowledge is lacking in the human oral microbiota. The ultimate goal of this proposal is to develop alternative therapeutics to combat bacteria-associated oral diseases and, the immediate goal is to establish a phage infection model system to acquire the knowledge of phage infection and replication in oral pathogens. A. actinomycetemcomitans develops mechanisms to resist serum killing and survives in the inflammatory periodontal pockets and the blood. Both environments provide serum as the main nutrient source for bacterial growth. However, our data indicated that selective clinical strains of this microorganism clearly demonstrated a ?bimodal? growth pattern only in the presence of human serum, and the secondary increase of optical density was due to bacterial lysis, therefore were grouped as ?serum-sensitive? strains. This novel finding leaded to the discovery of a 44kb Aggregatibacter phage S1249 in the serum-sensitive strain and, 20% of the phage genes were up regulated over 10-fold in human serum comparing to other growth media. We propose that human serum induces the replication of this phage resulting in bacterial killing and, therefore phage S1249 is a potential candidate to be used for studying phage replication and regulation in this oral pathogen. We will start our investigation by accomplishing two specific aims in this proposal: Firstly, we will develop a phage infection model system by deletion of the prophage DNA from the bacterial chromosome to cure this phage, following by re-infection with the same phage. Phage induction and bacterial fitness in human serum will be characterized and compared between the wild type and its isogenic cured strain at the RNA and protein levels. Secondly, we will determinate the infection specificity of phage S1249 by using this phage to infect other serum-resistant strains to evaluate the growth fitness of those newly infected strains in human serum. This physiologically relevant model system simulates periodontal environment and will provide fundamental tools and knowledge for our future investigations, which includes: identification of bacterial surface receptors for the phage absorption and, determination of human serum components that trigger phage induction. Ultimately, the knowledge we gain will unravel phage infection and replication regulation by serum in this oral pathogen.